Studies of Functional Characterization and Secretion Mechanism of Low-Molecule-Weight bacteriocin from Pectobacterium carotovorum subsp. carotovorum

Pectobacterium carotovorum subsp. carotovorum 是一株屬於腸道菌科的革蘭氏陰性菌，舊稱為伊文氏桿菌 (Erwinia)。這種革蘭氏陰性菌偏好寄生於植物，它經常造成經濟作物損傷。被此細菌寄生的植物在高溫潮溼的環境下會產生根部腐爛的疾病，所以這種有名的植物致病菌一直以來是許多科學家致力研究重點之一。現行針對此種植物致病菌經常會採用以化學物質為主成分的抗菌藥劑進行防疫，但這些化學試劑並不是一種很有效的方法，原因是化學試劑的影響是全面的並沒有針對性。這些結果可能會影響到植栽區域的正常生態鏈，以及無法避免的環境污染。 無論是革蘭氏陽性或者是革蘭氏陰性菌都會分泌許多胞外蛋白質性物質，其中一種胞外蛋白質稱為細菌素。細菌素是一種由蛋白質組成的抗菌性毒性物質。此類毒性物質被生產者利用來抑制其他菌株的生存。然而這種蛋白質性的毒性物對於其所抑制菌株的種類是相當有限且有其特異性。換句話說此類具有攻擊特異性的蛋白質性物質或許是一種可以利用來有效防疫 Pectobacterium 所造成的損失。同時蛋白質性物質不易造成環境污染，故或許可以利用細菌素來做為一種不錯的生物性抗菌試劑。目前已經有許多文獻探討過這種蛋白質性物質，例如大腸桿菌所產生的大腸桿菌素以及綠膿桿菌所產生的綠膿桿菌素都是相當著名的細菌素。本篇論文即介紹我們在伊文氏桿菌也成功得選殖出低蛋白分子量的細菌素基因，並且將此類低分子量細菌素命名為 Carocin。同時我們也發現主司 Carocin 分泌機制的分泌系統為革蘭氏陰性菌上常見的第三類型分泌系統。雖然許多革蘭氏陰性菌皆保有此種分泌系統，但是過去卻沒有相關文獻證實過第三類分泌系統會參與細菌素的分泌。 本篇論文利用細菌的接合生殖將帶有抗藥性基因的 DNA 分子送入待研究的細菌素生產者細胞內進行非特定的基因阻斷。在此處我們選用本身帶有的轉位子 Tn5 的菌株 E. coli 1830 來當做接合生殖實驗的提供者，而待研究的伊文氏桿菌則為轉位子的接收者，期望能得到帶有細菌素相關基因被轉位子阻斷的突變株。另外對於特定基因的阻斷，則是利用同質互換的實驗方法來得到特定基因阻斷的突變株。這些被阻斷的基因則是用不對稱交聯聚合酵素鏈鎖反應 （Thermal asymmetric interlaced PCR） 解析其 DNA 序列。並且同時利用南方點墨法來驗證其結果，而南方點墨法亦利用於製備 Genomic DNA library，幫助我們可以得到直接由染色體上截取特定區域的 DNA 分子。北方點墨法與 RNA 逆轉錄實驗則是用來觀察實驗中突變株細胞內的基因轉錄情形。 藉由轉位子 Tn5 阻斷基因的實驗方法，我們找到並且已經發表了兩個由伊文氏桿菌所生產的低分子量細菌素。 Carocin S1 為第一個發表的低分子量細菌素，它是一種由 Pcc 菌株 H-rif-8-6 所產生，可將 DNA 水解的核酸水解型細菌素。另外一個由 F-rif-18 產生的細菌素則命名為 Carocin S2。 經由核酸水解實驗， Carocin S2 被證實為一種可以水解 RNA 分子的細菌素，但令我們感興趣的是此種細菌素不但可以水解相對分子量最大的核糖體 RNA 也可水解其它小分量的 RNA。我們推測不論是 Carocin S1，亦或是 Carocin S2 在感染其他細菌細胞後，皆會攻擊它們所辨識的 DNA 或 RNA 分子，並進行水解破壞，使基因表現不正常，進而導致細菌細胞凋亡。而同時生產者本身亦會表現免疫蛋白來保護自己免於被自己生產的細菌素 Carocin 所傷害。另外，我們也發現伊文氏桿菌是藉由第三類分泌系統的鞭毛型構造，將毒性蛋白 Carocin 運輸至胞外，進而攻擊其它細菌細胞。 本篇論文將闡述我們實驗室發現了兩個由 Pectobacterium 產生的核酸水解型的低分子量細菌素 (Carocin S1 與 Carocin S2)，此種細菌素經常需要 UV 照射誘導使其表現。另外我們也證實此類細菌素是由鞭毛型第三類型分泌系統來輔助其分泌。Pectobacterium carotovorum subsp. carotovorum is a Gram-negative, phytoparasitic enterobacterium. It is also a well-known phytopathogen causing soft-rot disease of many economic crops. Chemical bactericidal is a current agent used against the disease but unavoidably causes the environmental contamination. Bacteriocins are endogenous, antimicrobial and toxic proteins, which are usually produced by Gram-positive and Gram-negative bacteria. However, the proteinaceous toxins have narrow spectrum to inhibit growth of the related bacteria; that is, bacteriocins would be a eco-friendly and efficient choice to prevent pathogen that causes the economic damage. While bacteriocins have been extensively investigated in many Gram-negative bacteria such as pyocin of Pseudomonas and colicin of Escherichia coli, they have been relatively unexplored in Pectobacterium species. The dissertation described that the Pcc strain also produces bacteriocin, designated as Carocin. Furthermore, we showed the secretion dependency of Carocin from Pcc strain used the type III secretion system, whereas little is shown about the relationship between them. In this study, the bacterial conjugation and the homologous replacement method were performed to translocate a linear construct harboring the antibiotic-resistance gene into the carocin-producing cell, resulting in the carocin-related null alleles. E. coli 1830 strain harboring transposon Tn5 was used as donors while the recipients were Pcc strains. In contrast to the conjugation, the homologous replacement method was used to knock out the specific target gene in genomic DNA. By using the thermal asymmetric interlaced PCR, the interrupted DNA sequence of the carocin-related null allele was determined. The southern blotting was used to confirm the result of mutation, moreover, the method would be used to prepare the genomic DNA library from which the native carocin-contained DNA was obtained. Additionally, transcriptional analysis was carried out by the Northern blotting and the reverse transcription PCR. These methods provide more information concerning with the carocins. Consequently, it was found that the Carocin S1 was produced from a Pcc strain H-rif-8-6. Carocin S1 has nucleotidase activity against DNA molecule. This is the first bacteriocin determining from Pectobacterium. Subsequently, Carocin S2 was characterized from Pcc strain F-rif-18, which was a RNase type bacteriocin. In the in vitro RNA degradation assay, Carocin S2 would hydrolyze not only the large molecule of ribosomal RNA but small RNA molecules. We suggested that both Carocin S1 and Carocin S2 kill those susceptive cells by exhausting their supply of DNA or RNA respectively, and then leading to inactivation of physiological biosynthesis. The two producers also have expression of the cognate immunity proteins which protect themselves from the specific damage of their toxic Carocins. Eventually, we established that Carocin protein might be secreted though the flagella that belongs type III secretion system. This secretion mechanism was different from those of previous reports of other bacteriocins. Here we showed the first two low-molecule-weight bacteriocins, Carocin S1 and Carocin S2, which are produced by Pectobacterium after UV irradiation. Furthermore, we found that the Carocin secretion is dependent on the type III secretion system integral to the bacterial flagellum, which this finding is irrelevant to the previous studies of bacteriocin secretion.Abstract 中文摘要.......................................................................................... i Abstract................................................................................................ iii Chapter 1 General introduction........................................................... 1 Features of Pectobacterium carotovorum subsp. carotovorum.............. 2 Features of bacteriocin........................................................................ 2 Induction of bacteriocin....................................................................... 4 Importation of bacteriocin.................................................................... 5 Killer domain of bacteriocin................................................................. 6 Secretion of bacteriocin......................................................................... 7 Summary............................................................................................... 10 Chapter 2 Cloning and Expression of the Erwinia carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1......................................................................................................... 11 Introduction........................................................................................... 12 Materials and methods.......................................................................... 12 Results................................................................................................... 18 Discussion.............................................................................................. 20 Figures................................................................................................... 23 Chapter 3 Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum............................. 33 Introduction............................................................................................ 34 Materials and methods................................................................................................. 34 Results................................................................................................... 43 Discussion.............................................................................................. 48 Figures................................................................................................... 51 Chapter 4 Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum............................................................................................... 67 Introduction........................................................................................... 68 Materials and methods.......................................................................... 68 Results................................................................................................... 76 Discussion.............................................................................................. 80 Figures................................................................................................... 83 Chapter 5 Conclusion............................................................................. 96 Conclusion.............................................................................................. 97 Figures...................................................................................................103 Tables Table 1. Bacteria and plasmids used in the study.................................105 Table 2. Primers used in this study.......................................................106 Table 3. The thermal processes of TAIL-PCR..........................................107 References.............................................................................................10

Gene Cloning and Analysis of Low-Molecular-Weight Bacteriocin Secretary Protein of Erwinia carotovora subsp. carotovora

Erwinia. carotovora subsp. carotovora是一種會造成許多植物根莖部腐爛的病原菌。大多作物常因感染此病原菌而失去經濟價值，但是到目前為止仍沒方法可以有效抑制此菌種造成的損害。由於此菌種會生產一至數種細菌素以抑制其他親屬相近的菌種生長，而被當作是一種有效的微生物農藥使用，但其細菌素相關研究仍然十分不足。 TH22-6 是一株被transposon Tn5 阻斷其低分子量細菌素生產相關基因的Erwinia. carotovora subsp. carotovora 野生株H-rif-8-6 的突變株。我們利用TAIL-PCR 的方法增幅並定序transposon Tn5 左右兩端向外延伸的DNA片段。此序列經過美國國家衛生研究院醫學圖書館的資料庫(NCBI)比對胺基酸序列(BLAST)，結果發現在此DNA片段與第三類型分泌系統的蛋白胺基酸序列有相當高的同質性。因此我們認為被Tn5 阻斷的基因為一種類似第三類分泌系統蛋白的基因。根據此比對結果我們推測Erwinia. carotovora subsp. carotovora可能經由類似第三類分泌系統的機制運送低分子量細菌素。 第三類分泌系統主要功能為分泌由病原菌產生的毒性蛋白，目前文獻指出其作用對象為動物或植物細胞。有趣的是在本文，我們卻發現低分子量細菌素可藉由類似第三類型分泌系統的機制分泌並感染其他菌種。希望此結果能幫助我們更加了解Erwinia carotovora subsp. carotovora 產生的低分子量細菌素其分泌系統作用的機制。Erwinia. carotovora subsp. carotovora is a phytopathogenic bacterium responsible for the soft-rot disease of many plants. It will decompose the crops, but there is no efficiently approach to bate the damages caused by the bacterium. Low-molecular-weight bacteriocin is an antibacterial substance which could be produced by Erwinia. carotovora subsp. carotovora, and inhibit the related strain growth. The bacteriocin could be used as effectively biopesticides, but the realtived study is still not enough. TH22-6 is a Transposon Tn5 insertional mutant strain from Erwinia. carotovora subsp. carotovora, it could not generate low-molecular-weight bacteriocin. We used TAIL-PCR method to amplify and analyze the DNA sequence of the insert end from transposon Tn5. The DNA sequence was compared with the BLAST of the National Center for Biotechnology Information server, then the result show that has high homology with the genes of secretary protein in bacterium, especially the type Ⅲ secretion system. According to the comparison, this result suggest the low-molecular- weight bacteriocin may be transport by the type Ⅲ-like secretion system. The function of type Ⅲ secretion system is usually the infection to the animal or the plant cell. In this study, it is interesting to find the relationship between low-molecular-weight bacteriocin and the type Ⅲ-like secretion system. The result may helpfully let we know the mechanism of low-molecular-weight bacteriocin and secretion system in Erwinia. carotovora subsp. carotovora.目次 第一章 緒論 …………………………………….………………………….....1 第二章 利用Transposon Tn5 阻斷E.C.C. 的低分子量細菌素生產相關基因 一. 目的……………………………………………………………..….8 二. 材料與方法……………………………………………………..….8 三. 結果……………………………………………………………….10 第三章 利用TAIL-PCR 的方法增幅並定序被Transposon Tn5阻斷的基因 一. 目的…………………………………………………………….…12 二. 材料與方法……………………………………………………….12 三. 結果……………………………………………………………….15 第四章 Erwinia. carotovora subsp. carotovora 低分子量細菌素分泌蛋白基因(BSP)的選殖 一. 目的…………………………………………………………….…21 二. 材料與方法……………………………………………………….21 三. 結果……………………………………………………………….24 第五章 BSP蛋白基因表現質體的構築與低分子量細菌素恢復實驗 一. 目的…………………………………………………………….…32 二. 材料與方法……………………………………………………….32 三. 結果……………………………………………………………….34 第六章 結論 ………………………………………………………………... 44 附錄………………………………………………………………………………55 參考文獻…………………………………………………………………………6